The transition to a dysfunction endothelial cell phenotype is regulated by multiple environmental factors, including both systemic risk factors (ex. oxidized LDL) and local blood flow patterns. My research suggests that local matrix composition is a novel regulator of endothelial cell dysfunction. Transitional matrix proteins (ex. fibronectin) accumulate in the subendothelial matrix early during atherogenesis and prime endothelial cells for dysfunction by enhancing flow-induced permeability and proinflammatory gene expression. In contrast, basement membrane proteins limit endothelial cell dysfunction. Multiple atherogenic stimuli, including disturbed flow and oxidized LDL, activate the Rac/cdc42 effector p21 activated kinase (PAK), and PAK inhibitors reduce endothelial permeability and proinflammatory responses both in vitro and at atherosclerosis- prone sites in vivo. Despite activation of upstream pathways, basement membrane proteins do not support PAK activation, suggesting that signals from the basement membrane inhibit PAK to limit endothelial cell dysfunction. As such, PAK activation in vivo is restricted to regions of transitional matrix deposition. Protein kinase A (PKA) phosphorylates and inhibits PAK in cells in suspension. In addition, PKA reduces both proinflammatory gene expression and endothelial permeability, suggesting PKA is a good candidate for matrix- specific PAK suppression. Preliminary data show that basement membrane proteins enhance flow-induced PKA activation, and inhibiting PKA in cells on basement membrane proteins is sufficient to restore flow- induced PAK activation and proinflammatory responses. These data lead us to hypothesize that basement membrane proteins utilize a PKA-dependent signal to inhibit PAK and limit endothelial cell dysfunction, whereas transitional matrix deposition primes endothelial cells to progress to a dysfunctional phenotype. The proposed work will test this hypothesis by determining the mechanisms by which matrix signaling modulates PKA activation (Aim 1) and by exploring the molecular mechanisms of PKA-dependent PAK inhibition (Aim 2). To illustrate the broad scope of this novel signaling axis across multiple atherogenic signals, we will determine how matrix composition and signaling through the PKA and PAK pathways affect oxidized LDL-induced endothelial cell dysfunction (Aim 3). This work will utilize a multifactorial approach to provide insight into the role of the endogenous basement membrane as a novel atheroprotective agent. PUBLIC HEALTH RELEVANCE: Atherosclerosis, a chronic inflammatory disease of the vessel wall, is the leading cause of death in developed countries. Our research suggests that changes in the local extracellular matrix may serve as a form of tissue memory. Normal components of the tissue matrix limit cellular responsiveness to transient injurious stimuli, while transitional matrix deposition in response to chronic stimuli enhances cellular responsiveness to propagate tissue remodeling. Understanding the molecular mechanisms by which matrix composition affects cell physiology could provide novel therapeutic targets to limit chronic inflammatory diseases, such as atherosclerosis and arthritis.